Fisetin inhibits patulin-induced cardiomyocyte apoptosis by regulating ROS/Grp78/Chop/Caspase-12

Fisetin inhibits patulin-induced cardiomyocyte apoptosis by regulating ROS/Grp78/Chop/Caspase-12 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Fisetin inhibits patulin-induced cardiomyocyte apoptosis by regulating ROS/Grp78/Chop/Caspase-12 Dongmei Xu, Baigang Zhang, Chenghui Huang, Jiao Lu, Yang Li, Binggang Fu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4839276/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objectives of the Study Fisetin (FIS) has a good protective effect on the heart. However, fisetin in regulating the role of the myocardial injury induced by patulin (PAT) is not clear. The aim of this study is to investigate the possible mechanism of fisetin in attenuating patulin induced myocardial injury. Materials and Methods Cardiomyocytes were treated with 25μM PAT to set the control group, FIS only group, PAT only group and PAT-FIS addition group. LDH activity, SOD content, and MDA content were evaluated using kits. ROS levels were determined by measuring the intensity of fluorescence. Mitochondrial membrane potential was detected by JC-1 dye staining. The protein expressions of Grp78, Chop and Caspase-12 were detected by Western blot. Results In PAT-FIS group, LDH release and MDA content decreased, but SOD content increased. Compared with the control group, the level of ROS in PAT group increased more than 10 times. The level of ROS in the PAT-FIS group was still higher than that in the control group, but it was significantly lower than that in the PAT group. The proportion of red fluorescence in the mitochondrial membrane potential of cardiomyocytes increased from 75% to 85% in the PAT-FIS group. PAT up-regulated the expression of Chop, Grp78 and Caspase-12 proteins, while the overexpression of Chop, Grp78 and Caspase-12 proteins was inhibited after pretreatment with FIS and PAT . Conclusion Our findings suggest that FIS inhibits PAT-induced cardiomyocyte apoptosis by regulating ROS/Grp78/Chop/Caspase-12 signaling. patulin fisetin myocardial damage Grp78 Chop Caspase-12 Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Fisetin(C 15 H 10 O 6 , FIS) is a naturally occurring flavonoid, found in a variety of fruits (mangoes, apples, strawberries, kiwifruit and grapes), vegetables (cucumbers, tomatoes and Onions), nuts and wine [ 1 ]. Studies have shown that FIS has many beneficial properties, including anti-cancer, anti-clotting, anti-inflammatory and antioxidant effects[ 2 ]. We have previously shown that FIS can inhibit PAT-induced cardiomyocyte apoptosis by inhibiting Caspase3/8/9, P53, and Bcl-2/Bax targets [ 3 ]. In addition, FIS has been shown to inhibit ROS/ER stress-mediated inflammatory responses, helping to improve cardiac damage induced by metabolic stimuli in vivo and in vitro [ 4 ]. FIS alleviates hypertension-related cardiac hypertrophy in H9c2 cells and spontaneously hypertensive rats [ 5 ]. FIS also plays a cardioprotective role in ameliorating oxidative stress, inflammation, and apoptosis in diabetic cardiomyopathy induced by HFD or streptozotocin (STZ) [ 6 ] [ 7 ]. Patulin (PAT) is a mycotoxin produced primarily by Penicillium expansum. Patulin was first found in mouldy apples and apple juice, and mainly contaminated fruits and their products, especially apples, hawthorns, pears, tomatoes, apple juice and hawthorn slices [ 8 ]. In addition, patulin contamination also exists in some Chinese medicinal materials (codonopsis, astragalus, etc.) and feed [ 9 ]. Toxicological studies of PAT have shown that ingestion of PAT affects a variety of organs such as the brain, liver and kidney in experimental animals [ 10 ] [ 11 ] [ 12 ]. Long-term exposure also has neurotoxic, immunotoxic, genotoxic and teratogenic effects [ 13 ]. After entering cardiomyocytes, PAT induces oxidative stress and inflammatory response of cardiomyocytes, resulting in apoptosis of cardiomyocytes, myocardial toxicity and cardiac injury [ 14 ]. As mentioned above, PAT can induce severe toxicity, and FIS is a promising natural extract for heart protection. However, as far as we know, the inhibitory mechanism of FIS on PAT is not very clear. Therefore, in this study, the contents of LDH, SOD, MDA, ROS and mitochondrial membrane potential in H9c2 cardiomyocytes were measured. Finally, the possible detoxification mechanism of FIS was explored by detecting the expressions of Grp78, Chop and Caspase-12 proteins in H9c2 cardiomyocytes. 2. Materials and Methods 2.1 Materials and Reagents Patulin (CAS: 149-29-1, purity ≥ 99%) was purchased from Sigma-Aldrich (Shanghai, China). Fisetin (CAS: 528-48-3, purity ≥ 98%) was purchased from AbMole. BCA protein detection kit, IP cell lysis buffer, phenylmethyl sulfonyl fluoride (PMSF) were obtained from Beyotime (Shanghai, China). Polyvinylidene fluoride (PVDF) membranes with an average pore size of 0.45µm were supplied by Millipore (St. Louis, USA). LDH test kit, MDA test kit, acrylamide, double acrylamide, triple, glycine and ammonium persulfate (AP) solution were obtained from Solarbio (Beijing, China). SOD detection kit and ROS detection kit were obtained from Nanjing Jiancheng Biological Company. The JC-1 mitochondrial membrane Potential (MMP) test kit was purchased from US EVERBRIGHT INC. SDS-PAGE Loading buffer (5x) was purchased from CWBIO (Beijing, China). All of the above chemicals are standard analytical grade or higher. 2.2 The contents of LDH, SOD and MDA in cells were detected H9c2 cardiomyocytes were purchased from the Laboratory of the Chinese Academy of Sciences in Shanghai. Cells were routinely cultured with 10%FBS and 1% penicillin-streptomycin in an incubator at 37°C and 5%CO 2 . According to our previous study [ 3 ], cardiomyocytes were treated with 25µM PAT in a control group, FIS only group, PAT only group, and PAT-FIS addition group. After cell culture and treatment, refer to the instructions of LDH test kit, SOD test kit and MDA test kit respectively for operation. 2.3 H9c2 cardiomyocytes ROS content determination The final concentration of DCFH-DA was 10 µM after 1000 times dilution in serum-free medium. Dilute DCFH-DA was added into the cells and incubated at 37℃ for 20 min. After 20 min, DCFH-DA was absorbed and the cells were washed without serum culture solution, and this was repeated three times. Then, the cells were collected and transferred into the flow tube. The fluorescence intensity before and after stimulation was detected in real time by cytometry at excitation wavelength of 488 nm and emission wavelength of 525 nm. 2.4 Mitochondrial membrane potential (MMP) determination Take an appropriate amount of JC-1 (200×) and dilute JC-1 by adding 8 mL of ultra-pure water every 50 µL JC-1 (200×). Fully dissolve and mix JC-1 by violent shock. Then add 2 mL JC-1 dyeing buffer (5 ×), and mix it into JC-1 dyeing working solution. The treated cells were removed from the incubator, digested into a centrifuge tube and centrifuged at 400 g for 5 min. After the supernatant was removed, the culture solution was re-suspended, and JC-1 working solution was added according to the ratio of 1:1 between the culture solution and JC-1 working solution, and incubated at 37℃ for 20 min. After incubation at 37℃, centrifuge 600 g at 4℃ for 3 ~ 4 min and discard the supernatant. The cells were washed twice with JC-1 buffer, then re-suspended with JC-1 buffer and detected by flow cytometry. The data is post-processed by Flow Jo software. 2.5 Western blot analysis Protein was extracted using RIPA lysis and Extraction buffer (Solarbio). The protein content was determined using the BCA protein assay kit (Beyotime). The extracted proteins (40µg) were placed in a 10–12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) system and then transferred to PVDF membranes (Millipore Company, USA). Next, close the membrane with 5% skim milk for 1 hour. They were incubated with primary antibodies at 4℃ overnight. Wash the PVDF membrane. After that, the cells were incubated with the second antibody for 1.5 hours. After the second antibody incubation, PVDF membrane was removed and washed for 3 times. An appropriate amount of luminescent liquid was added to the PVDF membrane, and then placed in the gel imaging system, exposed, and photographed. Image J software was used for gray scale analysis. The ratio of target band to internal reference band was taken as the result, the ratio of samples in each treatment group to blank control indicated the protein level, and the value of control group was set to 1. 2.6 Statistical analysis The result is expressed as "mean ± standard deviation". The Graphpad prism 9.5 software was used for significance analysis, and P < 0.05 indicated a significant difference, p < 0.01, denoted by "**". 3. Results 3.1 FIS inhibits oxidative stress induced by PAT Compared with Control, LDH release in PAT group was increased by 2.5 times (p < 0.05). Compared with PAT group, LDH release was significantly reduced in FIS+PAT group (p < 0.001) (Fig.1A). SOD activity in FIS group (20Μm FIS and 30μM FIS) had no significant change, but SOD activity in PAT group was significantly decreased compared with blank group (p < 0.001), and SOD activity in PAT group could only reach 1/2 of that in blank group (Fig.1B). Compared with PAT group, SOD content in FIS treatment group (25μM PAT+ 20μM FIS and 25μM PAT+ 30μM FIS) was significantly increased, with statistical significance. There was no significant difference in MDA content between control group and FIS group. MDA content in 25μM PAT group was significantly increased, reaching 6 times of that in control group. After FIS treatment, MDA content decreased significantly compared with 25μM PAT group, that is, MDA content in PAT +FIS group decreased by 4 times as much as that in control group (Fig.1C). These results indicated that cells were more sensitive to PAT stimulation, and oxidative damage occurred in cells, which seriously affected lipid oxidation and increased MDA content. However, FIS treatment decreased MDA content. 3.2 FIS pretreatment reduced PAT-induced ROS levels There was no significant difference in ROS intensity between 20μM FIS and 30μM FIS groups compared with control group (Fig.2). Compared with the control group, the fluorescence intensity and ROS level in PAT group were increased by more than 10 times. After FIS pretreatment, although the ROS level was still higher than that of the control group, the ROS level was significantly lower than that of the PAT group. Therefore, we can boldly speculate that PAT can cause cells to produce excess and harmful ROS, and then induce cell oxidative damage, leading to apoptosis and necrosis; However, FIS treatment can effectively inhibit the toxic effect of PAT and enhance the ability of cells to resist foreign stimuli. 3.3 The effect of PAT on mitochondrial membrane potential of cardiomyocytes decreased after FIS treatment The results were analyzed by a 2-dimensional scatter plot, which was divided into four quadrants by a cross gate. The upper right quadrant (UR) shows that JC-1 exists in the cell in polymer form and fluoresces red. The lower right quadrant indicates that JC-1 is present in cells as a monomer and emits green fluorescence. When the mitochondrial membrane potential is reduced, JC-1 switches from red to green fluorescence, a hallmark signal of early apoptosis. After PAT treatment, the mitochondrial membrane permeability transport pore (MPTP) of H9c2 cardiomyocytes was opened, and the internal and external charge of mitochondria was disordered, which was manifested as a significant increase in green fluorescence in the PAT group compared with the control group (Fig.3). However, FIS pretreatment reduced the effect of PAT on mitochondrial membrane potential, and the proportion of red fluorescence in the mitochondrial membrane potential increased from 75% in PAT group to 85% in FIS group. 3.4 FIS inhibits PAT-induced cardiomyocyte apoptosis by regulating Grp78/Chop/Caspase-12 After ER stress is activated, Grp78 is released as a key ER stress sensor [15]. Severe ER stress can promote the expression of pro-apoptotic proteins, such as Chop [16]. Finally, cells undergo apoptotic cell death under stimulating conditions [15]. Caspase-12, Grp78 and Chop, as important markers of endoplasmic reticulum stress-mediated apoptosis, play a key role in the apoptosis process [17]. The expressions of apoptosis-related proteins Caspase-12, Chop and Grp78 were detected by Western blotting. The ratio of apoptosis-related proteins to β-actin was quantified by ImageJ software. As shown in Figure 4, PAT up-regulated the expression levels of Chop, Grp78, and Caspase-12 proteins; while adding PAT after FIS pretreatment inhibited the overexpression of Chop, Grp78, and Caspase-12 proteins compared with adding PAT only (p < 0.001). 4. Discussion Apoptosis is an active process of cell death, involving two pathways: mitochondrial pathway and membrane receptor pathway [ 18 ]. The endoplasmic reticulum plays an important role in a variety of cellular processes required for cell survival and normal cell function, including intracellular calcium homeostasis, protein secretion and folding of secreted proteins, lipid biosynthesis, and it is involved in the intrinsic pathway of apoptosis [ 19 ]. At rest, Grp78 binds to an electrorheological membrane protein. However, under stimulated conditions, Grp78 will be released from endoplasmic reticulum proteins, resulting in homologous oligomerization of protein kinase R-like endoplasmic reticulum kinase (PERK), inositol dependent enzyme 1 (IRE1), and a large number of unfolded proteins accumulate in the endoplasmic reticulum [ 20 ]. Activation of the transcription factor C/EBP homologous protein (CHOP) triggers the endoplasmic reticulum to cause pro-apoptotic signaling, thereby promoting the activation of pro-apoptotic proteins and leading to apoptosis [ 21 ]. Endoplasmic reticulum stress may also lead to oxidative stress, resulting in toxic accumulation of ROS within cells [ 22 ]. ROS play a key role in processes such as cell signaling, stress response and cell death [ 23 ] [ 24 ]. There is a close association between mitochondrial membrane potential and ROS [ 25 ]. When cells are under oxidative stress, a large number of reactive oxygen species (ROS) are produced in mitochondria, which will lead to a decrease in mitochondrial membrane potential. At the same time, the decrease of mitochondrial membrane potential further increases ROS production. In our study, PAT caused oxidative stress in cardiomyocytes with increased ROS. And FIS can significantly inhibit oxidative stress caused by PAT, and inhibiting ROS generation, make PAT has less of an effect on myocardial cell mitochondrial membrane potential. PAT treated H9c2 cardiomyocytes showed significantly increased protein expressions of Grp78 and Chop. FIS prevented the pro-apoptotic process by down-regulating Grp78 and Chop, and partially protected H9c2 cardiomyocytes. WB results showed that FIS could significantly down-regulate the overexpression of Caspase-12 protein induced by PAT and block the transmission of apoptosis signal. Caspase-12 is located in ER and is considered to be a specific apoptotic mediator in the ERS apoptosis pathway [ 26 ]. In the ERS state, the increase of intracellular calcium ions leads to cytoplasmic activation of calpsin, and the precursor of Caspase-12 located on the ER membrane is cut for activation and release into the cytoplasm [ 27 ]. After Caspase-12 is activated, Caspase-9 is activated, and Caspase-9 activates downstream Caspase-3, initiating the classical apoptotic pathway and ultimately leading to apoptosis [ 28 ]. 5. Conclusion In conclusion, there are two main findings from the current study. Firstly, we demonstrated that FIS treatment could alter PAT-induced oxidative stress, ROS content and MMP in cardiomyocytes. Secondly, we detected the protein expression of Caspase12, Chop, and Grp78 to confirm the cardioprotective effect of FIS treatment. Declarations Compliance with Ethical Standards Funding: This work was supported by the National Natural Science Foundation of China (31760495); the Nature Fund of Gansu Province(18JR3RA136); Qingdao Institute of Technology Field of Scientific Research Projects. Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors. Ackonwledgements This work was supported by the National Natural Science Foundation of China (31760495); the Nature Fund of Gansu Province(18JR3RA136); Qingdao Institute of Technology Field of Scientific Research Projects. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions All authors contributed to the study conception and design. Baigang Zhang : Determine the subject, Formal analysis, Investigation, Methodology, Validation, Visualization, Funding support. Dongmei Xu : Determine the subject, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing-review & editing. Chenghui Huang : Determine the subject, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing-review & editing. Lu Jiao : Formal analysis, Investigation, Methodology, Software， Investigation, Methodology, Validation, Writing-original draft, Writing - review & editing. Yang Li : Methodology, Visualization, Formal analysis, Software, Investigation, Project administration. Binggang Fu : Formal analysis, investigation, visualization, software, verification. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. References G. Grynkiewicz and O. M. Demchuk, "New Perspectives for Fisetin," (in English), Frontiers in Chemistry, Review vol. 7, 2019-October-30 2019. L. Long et al. , "Protective effects of fisetin against myocardial ischemia/reperfusion injury," (in eng), Experimental and Therapeutic Medicine, vol. 19, no. 5, pp. 3177–3188, 2020. D. Xu, B. Zhang, C. Huang, J. Lu, Y. Li, and B. Fu, "Effect and mechanism of Fisetin on myocardial damage induced by Patulin," (in eng), Molecular Biology Reports, vol. 50, no. 8, pp. 6579-6589, 2023. C.-X. Ge et al. , "Endoplasmic reticulum stress-induced iRhom2 up-regulation promotes macrophage-regulated cardiac inflammation and lipid deposition in high fat diet (HFD)-challenged mice: Intervention of fisetin and metformin," Free Radical Biology and Medicine, vol. 141, pp. 67-83, 2019/09/01/ 2019. K.-H. Lin et al. , "Bioactive flavone fisetin attenuates hypertension associated cardiac hypertrophy in H9c2 cells and in spontaneously hypertension rats," Journal of Functional Foods, vol. 52, pp. 212-218, 2019/01/01/ 2019. L.-F. Hu et al. , "Oral flavonoid fisetin treatment protects against prolonged high-fat-diet-induced cardiac dysfunction by regulation of multicombined signaling," The Journal of Nutritional Biochemistry, vol. 77, p. 108253, 2020/03/01/ 2020. O. Y. Althunibat, A. M. Al Hroob, M. H. Abukhalil, M. O. Germoush, M. Bin-Jumah, and A. M. Mahmoud, "Fisetin ameliorates oxidative stress, inflammation and apoptosis in diabetic cardiomyopathy," Life Sciences, vol. 221, pp. 83-92, 2019/03/15/ 2019. M. Xing, Y. Chen, B. Li, and S. Tian, "Characterization of a short-chain dehydrogenase/reductase and its function in patulin biodegradation in apple juice," Food Chemistry, vol. 348, p. 129046, 2021/06/30/ 2021. M. A. Al-Hazmi, "Patulin in apple juice and its risk assessments on albino mice," Toxicology and Industrial Health, vol. 30, no. 6, pp. 534-545, 2014. C. Wei et al. , "Progress in the distribution, toxicity, control, and detoxification of patulin: A review," Toxicon, vol. 184, pp. 83-93, 2020/09/01/ 2020. B. Zhang, C. Huang, Q. Lu, H. Liang, J. Li, and D. Xu, "Involvement of caspase in patulin-induced hepatotoxicity in vitro and in vivo," Toxicon, vol. 206, pp. 64-73, 2022/01/30/ 2022. O. Puel, P. Galtier, and I. P. Oswald, "Biosynthesis and Toxicological Effects of Patulin," Toxins , vol. 2, no. 4 , pp. 613-631. doi: 10.3390/toxins2040613 W.-L. Lei et al. , "Toxic effects of patulin on mouse oocytes and its possible mechanisms," Toxicology, vol. 464, p. 153013, 2021/12/01/ 2021. B. Zhang et al. , "Cardiotoxicity of patulin was found in H9c2 cells," Toxicon, vol. 207, pp. 21-30, 2022/02/01/ 2022. I. M. Ibrahim, D. H. Abdelmalek, and A. A. Elfiky, "GRP78: A cell's response to stress," Life Sciences, vol. 226, pp. 156-163, 2019/06/01/ 2019. T. Verfaillie, A. D. Garg, and P. Agostinis, "Targeting ER stress induced apoptosis and inflammation in cancer," Cancer Letters, vol. 332, no. 2, pp. 249-264, 2013/05/28/ 2013. M. Zhong, Z. Wu, Z. Chen, Q. Ren, and J. Zhou, "Advances in the interaction between endoplasmic reticulum stress and osteoporosis," Biomedicine & Pharmacotherapy, vol. 165, p. 115134, 2023/09/01/ 2023. Z. Jin and W. S. El-Deiry, "Overview of cell death signaling pathways," Cancer Biology & Therapy, vol. 4, no. 2, pp. 147-171, 2005/02/02 2005. T. Anelli and R. Sitia, "Protein quality control in the early secretory pathway," The EMBO Journal, vol. 27, no. 2, pp. 315-327, 2008/01/23 2008. A. S. Lee, "The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress," (in eng), Methods, vol. 35, no. 4, pp. 373-81, 2005. N. T. Sprenkle, S. G. Sims, C. L. Sánchez, and G. P. Meares, "Endoplasmic reticulum stress and inflammation in the central nervous system," Molecular Neurodegeneration, vol. 12, no. 1, p. 42, 2017/05/25 2017. S. Chakravarthi, C. E. Jessop, and N. J. Bulleid, "The role of glutathione in disulphide bond formation and endoplasmic‐reticulum‐generated oxidative stress," EMBO reports, vol. 7, no. 3, pp. 271-275-275, 2006/03/01 2006. I. B. Perez and P. J. Brown, "The role of ROS signaling in cross-tolerance: from model to crop," (in English), Frontiers in Plant Science, Mini Review vol. 5, 2014-December-23 2014. Y. Zhang, S. Choksi, K. Chen, Y. Pobezinskaya, I. Linnoila, and Z.-G. Liu, "ROS play a critical role in the differentiation of alternatively activated macrophages and the occurrence of tumor-associated macrophages," Cell Research, vol. 23, no. 7, pp. 898-914, 2013. Y. Che, Y. Tian, R. Chen, L. Xia, F. Liu, and Z. Su, "IL-22 ameliorated cardiomyocyte apoptosis in cardiac ischemia/reperfusion injury by blocking mitochondrial membrane potential decrease, inhibiting ROS and cytochrome C," Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, vol. 1867, no. 9, 2021. Q. Zhang et al. , "Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress," Apoptosis, vol. 21, no. 4, pp. 432-442, 2016/04/01 2016. Y. Tian, L. Wang, Z. Qiu, Y. Xu, and R. Hua, "Autophagy triggers endoplasmic reticulum stress and C/EBP homologous protein-mediated apoptosis in OGD/R-treated neurons in a caspase-12-independent manner," (in eng), Journal of neurophysiology, vol. 126, no. 5, pp. 1740-1750, 2021/11// 2021. H. Wootz, I. Hansson, L. Korhonen, U. Näpänkangas, and D. Lindholm, "Caspase-12 cleavage and increased oxidative stress during motoneuron degeneration in transgenic mouse model of ALS," Biochemical and Biophysical Research Communications, vol. 322, no. 1, pp. 281-286, 2004/09/10/ 2004. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4839276","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":344825714,"identity":"024e72b9-9169-4b91-89ca-ac9e26d0a08b","order_by":0,"name":"Dongmei Xu","email":"","orcid":"","institution":"Qingdao Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Dongmei","middleName":"","lastName":"Xu","suffix":""},{"id":344825716,"identity":"84215e33-2ad8-4e8b-a05f-790b8f231c2f","order_by":1,"name":"Baigang Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYDACCSBmbGDgAXM+GNjYkaaFcUZBWjLRWsCAmefDIRgbN5Cf3fzs4dcddjLy7mcPv7YxOMDMwH746AZ8WhjnHDM3lj2TzGN4Ji/NOsfgDh8DT1raDXxamCUSzKQl25h5DBtyzIxzDJ4xM0jwmOHVwiaR/g2opZ7HsP+NmbGFwWHGBkJaeCRyzCQ/th3mkZfIMX7MQIwWCYmcMmnGM8d5DCTemDH2GKQlsxHyi/yM9G2SP3dU28v35xh/+PHHxo6f/fAxvFpAgBkUjwYHgP4C+46QchBg/AGyroGB+QMxqkfBKBgFo2DkAQDvNUbcG403FAAAAABJRU5ErkJggg==","orcid":"","institution":"Lanzhou university of technology","correspondingAuthor":true,"prefix":"","firstName":"Baigang","middleName":"","lastName":"Zhang","suffix":""},{"id":344825717,"identity":"6f8f1bbb-99df-47ef-843f-1794b6764674","order_by":2,"name":"Chenghui Huang","email":"","orcid":"","institution":"Lanzhou university of technology","correspondingAuthor":false,"prefix":"","firstName":"Chenghui","middleName":"","lastName":"Huang","suffix":""},{"id":344825718,"identity":"8d33b6ac-3fae-46e5-a4f3-da1566c9cc4e","order_by":3,"name":"Jiao Lu","email":"","orcid":"","institution":"Lanzhou university of technology","correspondingAuthor":false,"prefix":"","firstName":"Jiao","middleName":"","lastName":"Lu","suffix":""},{"id":344825719,"identity":"3194d879-2646-48e7-a18a-6e39060aa939","order_by":4,"name":"Yang Li","email":"","orcid":"","institution":"Lanzhou university of technology","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Li","suffix":""},{"id":344825720,"identity":"1103b533-797f-4570-91bc-cfe308bffd2b","order_by":5,"name":"Binggang Fu","email":"","orcid":"","institution":"Lanzhou university of technology","correspondingAuthor":false,"prefix":"","firstName":"Binggang","middleName":"","lastName":"Fu","suffix":""}],"badges":[],"createdAt":"2024-08-01 04:51:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4839276/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4839276/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63663608,"identity":"12535c7d-ef0f-4284-9fbb-c57afcd4de72","added_by":"auto","created_at":"2024-08-30 18:54:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":64731,"visible":true,"origin":"","legend":"\u003cp\u003eFIS significantly attenuated PAT-induced oxidative stress. Using the kit to detect LDH release(Fig.1A), MDA content(Fig.1C) and SOD content(Fig.1B), all the results are expressed in average ±SD.(***p\u0026lt;0.001 vs control, ##p\u0026lt;0.01 vs 25μM PAT, ###p\u0026lt;0.001 vs 25μM PAT)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4839276/v1/db299aac8ebb2007b24f6878.png"},{"id":63663611,"identity":"4e549f4c-a7f2-4608-b2b7-85f4ecf073c0","added_by":"auto","created_at":"2024-08-30 18:54:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":20854,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different dosing methods on ROS content in H9c2 cells\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4839276/v1/bf0a3dcad92f29ff90cea818.png"},{"id":63663610,"identity":"381a9ed7-7e39-4fe4-8412-a25fff980297","added_by":"auto","created_at":"2024-08-30 18:54:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":405129,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different drug addition methods on mitochondrial membrane potential of H9c2 cells\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4839276/v1/377b03451ec78a7789263d31.jpg"},{"id":63664142,"identity":"1627a164-c8f0-4a4a-8d10-40461213204e","added_by":"auto","created_at":"2024-08-30 19:02:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":120872,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of FIS on PAT-induced expression of apoptosis-related proteins in H9c2 cells\u003c/p\u003e\n\u003cp\u003e（***p\u0026lt;0.001 vs control, ###p\u0026lt;0.001 vs 25μM PAT, ##p\u0026lt;0.01 vs 25μM PAT, #p\u0026lt;0.05 vs 25μM PAT\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4839276/v1/c391a605f9a2c940c5da25a3.png"},{"id":63969623,"identity":"fb35a529-1f9f-494f-b6a5-bd6f62c97398","added_by":"auto","created_at":"2024-09-04 10:42:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1106258,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4839276/v1/267a67dd-9e7d-4be7-bafb-bd51c50991c1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eFisetin inhibits patulin-induced cardiomyocyte apoptosis by regulating ROS/Grp78/Chop/Caspase-12\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFisetin(C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e, FIS) is a naturally occurring flavonoid, found in a variety of fruits (mangoes, apples, strawberries, kiwifruit and grapes), vegetables (cucumbers, tomatoes and Onions), nuts and wine [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Studies have shown that FIS has many beneficial properties, including anti-cancer, anti-clotting, anti-inflammatory and antioxidant effects[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. We have previously shown that FIS can inhibit PAT-induced cardiomyocyte apoptosis by inhibiting Caspase3/8/9, P53, and Bcl-2/Bax targets [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In addition, FIS has been shown to inhibit ROS/ER stress-mediated inflammatory responses, helping to improve cardiac damage induced by metabolic stimuli in vivo and in vitro [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. FIS alleviates hypertension-related cardiac hypertrophy in H9c2 cells and spontaneously hypertensive rats [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. FIS also plays a cardioprotective role in ameliorating oxidative stress, inflammation, and apoptosis in diabetic cardiomyopathy induced by HFD or streptozotocin (STZ) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePatulin (PAT) is a mycotoxin produced primarily by Penicillium expansum. Patulin was first found in mouldy apples and apple juice, and mainly contaminated fruits and their products, especially apples, hawthorns, pears, tomatoes, apple juice and hawthorn slices [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In addition, patulin contamination also exists in some Chinese medicinal materials (codonopsis, astragalus, etc.) and feed [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Toxicological studies of PAT have shown that ingestion of PAT affects a variety of organs such as the brain, liver and kidney in experimental animals [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Long-term exposure also has neurotoxic, immunotoxic, genotoxic and teratogenic effects [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. After entering cardiomyocytes, PAT induces oxidative stress and inflammatory response of cardiomyocytes, resulting in apoptosis of cardiomyocytes, myocardial toxicity and cardiac injury [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs mentioned above, PAT can induce severe toxicity, and FIS is a promising natural extract for heart protection. However, as far as we know, the inhibitory mechanism of FIS on PAT is not very clear. Therefore, in this study, the contents of LDH, SOD, MDA, ROS and mitochondrial membrane potential in H9c2 cardiomyocytes were measured. Finally, the possible detoxification mechanism of FIS was explored by detecting the expressions of Grp78, Chop and Caspase-12 proteins in H9c2 cardiomyocytes.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials and Reagents\u003c/h2\u003e \u003cp\u003ePatulin (CAS: 149-29-1, purity\u0026thinsp;\u0026ge;\u0026thinsp;99%) was purchased from Sigma-Aldrich (Shanghai, China). Fisetin (CAS: 528-48-3, purity\u0026thinsp;\u0026ge;\u0026thinsp;98%) was purchased from AbMole. BCA protein detection kit, IP cell lysis buffer, phenylmethyl sulfonyl fluoride (PMSF) were obtained from Beyotime (Shanghai, China). Polyvinylidene fluoride (PVDF) membranes with an average pore size of 0.45\u0026micro;m were supplied by Millipore (St. Louis, USA). LDH test kit, MDA test kit, acrylamide, double acrylamide, triple, glycine and ammonium persulfate (AP) solution were obtained from Solarbio (Beijing, China). SOD detection kit and ROS detection kit were obtained from Nanjing Jiancheng Biological Company. The JC-1 mitochondrial membrane Potential (MMP) test kit was purchased from US EVERBRIGHT INC. SDS-PAGE Loading buffer (5x) was purchased from CWBIO (Beijing, China). All of the above chemicals are standard analytical grade or higher.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 The contents of LDH, SOD and MDA in cells were detected\u003c/h2\u003e \u003cp\u003eH9c2 cardiomyocytes were purchased from the Laboratory of the Chinese Academy of Sciences in Shanghai. Cells were routinely cultured with 10%FBS and 1% penicillin-streptomycin in an incubator at 37\u0026deg;C and 5%CO\u003csub\u003e2\u003c/sub\u003e. According to our previous study [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], cardiomyocytes were treated with 25\u0026micro;M PAT in a control group, FIS only group, PAT only group, and PAT-FIS addition group. After cell culture and treatment, refer to the instructions of LDH test kit, SOD test kit and MDA test kit respectively for operation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 H9c2 cardiomyocytes ROS content determination\u003c/h2\u003e \u003cp\u003eThe final concentration of DCFH-DA was 10 \u0026micro;M after 1000 times dilution in serum-free medium. Dilute DCFH-DA was added into the cells and incubated at 37℃ for 20 min. After 20 min, DCFH-DA was absorbed and the cells were washed without serum culture solution, and this was repeated three times. Then, the cells were collected and transferred into the flow tube. The fluorescence intensity before and after stimulation was detected in real time by cytometry at excitation wavelength of 488 nm and emission wavelength of 525 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Mitochondrial membrane potential (MMP) determination\u003c/h2\u003e \u003cp\u003eTake an appropriate amount of JC-1 (200\u0026times;) and dilute JC-1 by adding 8 mL of ultra-pure water every 50 \u0026micro;L JC-1 (200\u0026times;). Fully dissolve and mix JC-1 by violent shock. Then add 2 mL JC-1 dyeing buffer (5 \u0026times;), and mix it into JC-1 dyeing working solution. The treated cells were removed from the incubator, digested into a centrifuge tube and centrifuged at 400 g for 5 min. After the supernatant was removed, the culture solution was re-suspended, and JC-1 working solution was added according to the ratio of 1:1 between the culture solution and JC-1 working solution, and incubated at 37℃ for 20 min. After incubation at 37℃, centrifuge 600 g at 4℃ for 3\u0026thinsp;~\u0026thinsp;4 min and discard the supernatant. The cells were washed twice with JC-1 buffer, then re-suspended with JC-1 buffer and detected by flow cytometry. The data is post-processed by Flow Jo software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Western blot analysis\u003c/h2\u003e \u003cp\u003eProtein was extracted using RIPA lysis and Extraction buffer (Solarbio). The protein content was determined using the BCA protein assay kit (Beyotime). The extracted proteins (40\u0026micro;g) were placed in a 10\u0026ndash;12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) system and then transferred to PVDF membranes (Millipore Company, USA). Next, close the membrane with 5% skim milk for 1 hour. They were incubated with primary antibodies at 4℃ overnight. Wash the PVDF membrane. After that, the cells were incubated with the second antibody for 1.5 hours. After the second antibody incubation, PVDF membrane was removed and washed for 3 times. An appropriate amount of luminescent liquid was added to the PVDF membrane, and then placed in the gel imaging system, exposed, and photographed. Image J software was used for gray scale analysis. The ratio of target band to internal reference band was taken as the result, the ratio of samples in each treatment group to blank control indicated the protein level, and the value of control group was set to 1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe result is expressed as \"mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation\". The Graphpad prism 9.5 software was used for significance analysis, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated a significant difference, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, denoted by \"**\".\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003ch2\u003e3.1 FIS inhibits oxidative stress induced by PAT\u003c/h2\u003e\n\u003cp\u003eCompared with Control, LDH release in PAT group was increased by 2.5 times (p \u0026lt; 0.05). Compared with PAT group, LDH release was significantly reduced in FIS+PAT group (p \u0026lt; 0.001) (Fig.1A). SOD activity in FIS group (20\u0026Mu;m FIS and 30\u0026mu;M FIS) had no significant change, but SOD activity in PAT group was significantly decreased compared with blank group (p \u0026lt; 0.001), and SOD activity in PAT group could only reach 1/2 of that in blank group (Fig.1B). Compared with PAT group, SOD content in FIS treatment group (25\u0026mu;M PAT+ 20\u0026mu;M FIS and 25\u0026mu;M PAT+ 30\u0026mu;M FIS) was significantly increased, with statistical significance. There was no significant difference in MDA content between control group and FIS group. MDA content in 25\u0026mu;M PAT group was significantly increased, reaching 6 times of that in control group. After FIS treatment, MDA content decreased significantly compared with 25\u0026mu;M PAT group, that is, MDA content in PAT +FIS group decreased by 4 times as much as that in control group (Fig.1C). These results indicated that cells were more sensitive to PAT stimulation, and oxidative damage occurred in cells, which seriously affected lipid oxidation and increased MDA content. However, FIS treatment decreased MDA content.\u003c/p\u003e\n\u003ch2\u003e3.2 FIS pretreatment reduced PAT-induced ROS levels\u003c/h2\u003e\n\u003cp\u003eThere was no significant difference in ROS intensity between 20\u0026mu;M FIS and 30\u0026mu;M FIS groups compared with control group (Fig.2). Compared with the control group, the fluorescence intensity and ROS level in PAT group were increased by more than 10 times. After FIS pretreatment, although the ROS level was still higher than that of the control group, the ROS level was significantly lower than that of the PAT group. Therefore, we can boldly speculate that PAT can cause cells to produce excess and harmful ROS, and then induce cell oxidative damage, leading to apoptosis and necrosis; However, FIS treatment can effectively inhibit the toxic effect of PAT and enhance the ability of cells to resist foreign stimuli.\u003c/p\u003e\n\u003ch2\u003e3.3 The effect of PAT on mitochondrial membrane potential of cardiomyocytes decreased after FIS treatment\u003c/h2\u003e\n\u003cp\u003eThe results were analyzed by a 2-dimensional scatter plot, which was divided into four quadrants by a cross gate. The upper right quadrant (UR) shows that JC-1 exists in the cell in polymer form and fluoresces red. The lower right quadrant indicates that JC-1 is present in cells as a monomer and emits green fluorescence. When the mitochondrial membrane potential is reduced, JC-1 switches from red to green fluorescence, a hallmark signal of early apoptosis. After PAT treatment, the mitochondrial membrane permeability transport pore (MPTP) of H9c2 cardiomyocytes was opened, and the internal and external charge of mitochondria was disordered, which was manifested as a significant increase in green fluorescence in the PAT group compared with the control group (Fig.3). However, FIS pretreatment reduced the effect of PAT on mitochondrial membrane potential, and the proportion of red fluorescence in the mitochondrial membrane potential increased from 75% in PAT group to 85% in FIS group.\u003c/p\u003e\n\u003ch2\u003e3.4 FIS inhibits PAT-induced cardiomyocyte apoptosis by regulating Grp78/Chop/Caspase-12\u003c/h2\u003e\n\u003cp\u003eAfter ER stress is activated, Grp78 is released as a key ER stress sensor [15]. Severe ER stress can promote the expression of pro-apoptotic proteins, such as Chop [16]. Finally, cells undergo apoptotic cell death under stimulating conditions [15]. Caspase-12, Grp78 and Chop, as important markers of endoplasmic reticulum stress-mediated apoptosis, play a key role in the apoptosis process [17]. The expressions of apoptosis-related proteins Caspase-12, Chop and Grp78 were detected by Western blotting. The ratio of apoptosis-related proteins to \u0026beta;-actin was quantified by ImageJ software. As shown in Figure 4, PAT up-regulated the expression levels of Chop, Grp78, and Caspase-12 proteins; while adding PAT after FIS pretreatment inhibited the overexpression of Chop, Grp78, and Caspase-12 proteins compared with adding PAT only (p \u0026lt; 0.001).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eApoptosis is an active process of cell death, involving two pathways: mitochondrial pathway and membrane receptor pathway [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The endoplasmic reticulum plays an important role in a variety of cellular processes required for cell survival and normal cell function, including intracellular calcium homeostasis, protein secretion and folding of secreted proteins, lipid biosynthesis, and it is involved in the intrinsic pathway of apoptosis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. At rest, Grp78 binds to an electrorheological membrane protein. However, under stimulated conditions, Grp78 will be released from endoplasmic reticulum proteins, resulting in homologous oligomerization of protein kinase R-like endoplasmic reticulum kinase (PERK), inositol dependent enzyme 1 (IRE1), and a large number of unfolded proteins accumulate in the endoplasmic reticulum [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Activation of the transcription factor C/EBP homologous protein (CHOP) triggers the endoplasmic reticulum to cause pro-apoptotic signaling, thereby promoting the activation of pro-apoptotic proteins and leading to apoptosis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEndoplasmic reticulum stress may also lead to oxidative stress, resulting in toxic accumulation of ROS within cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. ROS play a key role in processes such as cell signaling, stress response and cell death [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. There is a close association between mitochondrial membrane potential and ROS [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. When cells are under oxidative stress, a large number of reactive oxygen species (ROS) are produced in mitochondria, which will lead to a decrease in mitochondrial membrane potential. At the same time, the decrease of mitochondrial membrane potential further increases ROS production. In our study, PAT caused oxidative stress in cardiomyocytes with increased ROS. And FIS can significantly inhibit oxidative stress caused by PAT, and inhibiting ROS generation, make PAT has less of an effect on myocardial cell mitochondrial membrane potential. PAT treated H9c2 cardiomyocytes showed significantly increased protein expressions of Grp78 and Chop. FIS prevented the pro-apoptotic process by down-regulating Grp78 and Chop, and partially protected H9c2 cardiomyocytes.\u003c/p\u003e \u003cp\u003eWB results showed that FIS could significantly down-regulate the overexpression of Caspase-12 protein induced by PAT and block the transmission of apoptosis signal. Caspase-12 is located in ER and is considered to be a specific apoptotic mediator in the ERS apoptosis pathway [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the ERS state, the increase of intracellular calcium ions leads to cytoplasmic activation of calpsin, and the precursor of Caspase-12 located on the ER membrane is cut for activation and release into the cytoplasm [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. After Caspase-12 is activated, Caspase-9 is activated, and Caspase-9 activates downstream Caspase-3, initiating the classical apoptotic pathway and ultimately leading to apoptosis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, there are two main findings from the current study. Firstly, we demonstrated that FIS treatment could alter PAT-induced oxidative stress, ROS content and MMP in cardiomyocytes. Secondly, we detected the protein expression of Caspase12, Chop, and Grp78 to confirm the cardioprotective effect of FIS treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the National Natural Science Foundation of China (31760495); the Nature Fund of Gansu Province(18JR3RA136); Qingdao Institute of Technology Field of Scientific Research Projects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e This article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAckonwledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China\u0026nbsp;(31760495); the Nature Fund of Gansu Province(18JR3RA136); Qingdao Institute of Technology Field of Scientific Research Projects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. \u003cstrong\u003eBaigang Zhang\u003c/strong\u003e: Determine the subject, Formal analysis, Investigation, Methodology, Validation, Visualization, Funding support. \u003cstrong\u003eDongmei Xu\u003c/strong\u003e: Determine the subject, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing-review \u0026amp; editing. \u003cstrong\u003eChenghui Huang\u003c/strong\u003e: Determine the subject, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing-review \u0026amp; editing. \u003cstrong\u003eLu Jiao\u003c/strong\u003e: Formal analysis, Investigation, Methodology, Software，\u0026nbsp;Investigation, Methodology, Validation, Writing-original draft, Writing - review \u0026amp; editing. \u003cstrong\u003eYang Li\u003c/strong\u003e: Methodology, Visualization, Formal analysis, Software, Investigation, Project administration. \u003cstrong\u003eBinggang Fu\u003c/strong\u003e: Formal analysis, investigation, visualization, software, verification. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eG. Grynkiewicz and O. M. Demchuk, \u0026quot;New Perspectives for Fisetin,\u0026quot; (in English), \u003cem\u003eFrontiers in Chemistry, \u003c/em\u003eReview vol. 7, 2019-October-30 2019.\u003c/li\u003e\n\u003cli\u003eL. Long\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Protective effects of fisetin against myocardial ischemia/reperfusion injury,\u0026quot; (in eng), \u003cem\u003eExperimental and Therapeutic Medicine, \u003c/em\u003evol. 19, no. 5, pp. 3177\u0026ndash;3188, 2020.\u003c/li\u003e\n\u003cli\u003eD. Xu, B. Zhang, C. Huang, J. Lu, Y. Li, and B. Fu, \u0026quot;Effect and mechanism of Fisetin on myocardial damage induced by Patulin,\u0026quot; (in eng), \u003cem\u003eMolecular Biology Reports, \u003c/em\u003evol. 50, no. 8, pp. 6579-6589, 2023.\u003c/li\u003e\n\u003cli\u003eC.-X. Ge\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Endoplasmic reticulum stress-induced iRhom2 up-regulation promotes macrophage-regulated cardiac inflammation and lipid deposition in high fat diet (HFD)-challenged mice: Intervention of fisetin and metformin,\u0026quot; \u003cem\u003eFree Radical Biology and Medicine, \u003c/em\u003evol. 141, pp. 67-83, 2019/09/01/ 2019.\u003c/li\u003e\n\u003cli\u003eK.-H. Lin\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Bioactive flavone fisetin attenuates hypertension associated cardiac hypertrophy in H9c2 cells and in spontaneously hypertension rats,\u0026quot; \u003cem\u003eJournal of Functional Foods, \u003c/em\u003evol. 52, pp. 212-218, 2019/01/01/ 2019.\u003c/li\u003e\n\u003cli\u003eL.-F. Hu\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Oral flavonoid fisetin treatment protects against prolonged high-fat-diet-induced cardiac dysfunction by regulation of multicombined signaling,\u0026quot; \u003cem\u003eThe Journal of Nutritional Biochemistry, \u003c/em\u003evol. 77, p. 108253, 2020/03/01/ 2020.\u003c/li\u003e\n\u003cli\u003eO. Y. Althunibat, A. M. Al Hroob, M. H. Abukhalil, M. O. Germoush, M. Bin-Jumah, and A. M. Mahmoud, \u0026quot;Fisetin ameliorates oxidative stress, inflammation and apoptosis in diabetic cardiomyopathy,\u0026quot; \u003cem\u003eLife Sciences, \u003c/em\u003evol. 221, pp. 83-92, 2019/03/15/ 2019.\u003c/li\u003e\n\u003cli\u003eM. Xing, Y. Chen, B. Li, and S. Tian, \u0026quot;Characterization of a short-chain dehydrogenase/reductase and its function in patulin biodegradation in apple juice,\u0026quot; \u003cem\u003eFood Chemistry, \u003c/em\u003evol. 348, p. 129046, 2021/06/30/ 2021.\u003c/li\u003e\n\u003cli\u003eM. A. Al-Hazmi, \u0026quot;Patulin in apple juice and its risk assessments on albino mice,\u0026quot; \u003cem\u003eToxicology and Industrial Health, \u003c/em\u003evol. 30, no. 6, pp. 534-545, 2014.\u003c/li\u003e\n\u003cli\u003eC. Wei\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Progress in the distribution, toxicity, control, and detoxification of patulin: A review,\u0026quot; \u003cem\u003eToxicon, \u003c/em\u003evol. 184, pp. 83-93, 2020/09/01/ 2020.\u003c/li\u003e\n\u003cli\u003eB. Zhang, C. Huang, Q. Lu, H. Liang, J. Li, and D. Xu, \u0026quot;Involvement of caspase in patulin-induced hepatotoxicity in vitro and in vivo,\u0026quot; \u003cem\u003eToxicon, \u003c/em\u003evol. 206, pp. 64-73, 2022/01/30/ 2022.\u003c/li\u003e\n\u003cli\u003eO. Puel, P. Galtier, and I. P. Oswald, \u0026quot;Biosynthesis and Toxicological Effects of Patulin,\u0026quot; \u003cem\u003eToxins\u003c/em\u003e, vol. 2, no. 4\u003cem\u003e, \u003c/em\u003epp. 613-631. doi: 10.3390/toxins2040613 \u003c/li\u003e\n\u003cli\u003eW.-L. Lei\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Toxic effects of patulin on mouse oocytes and its possible mechanisms,\u0026quot; \u003cem\u003eToxicology, \u003c/em\u003evol. 464, p. 153013, 2021/12/01/ 2021.\u003c/li\u003e\n\u003cli\u003eB. Zhang\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Cardiotoxicity of patulin was found in H9c2 cells,\u0026quot; \u003cem\u003eToxicon, \u003c/em\u003evol. 207, pp. 21-30, 2022/02/01/ 2022.\u003c/li\u003e\n\u003cli\u003eI. M. Ibrahim, D. H. Abdelmalek, and A. A. Elfiky, \u0026quot;GRP78: A cell\u0026apos;s response to stress,\u0026quot; \u003cem\u003eLife Sciences, \u003c/em\u003evol. 226, pp. 156-163, 2019/06/01/ 2019.\u003c/li\u003e\n\u003cli\u003eT. Verfaillie, A. D. Garg, and P. Agostinis, \u0026quot;Targeting ER stress induced apoptosis and inflammation in cancer,\u0026quot; \u003cem\u003eCancer Letters, \u003c/em\u003evol. 332, no. 2, pp. 249-264, 2013/05/28/ 2013.\u003c/li\u003e\n\u003cli\u003eM. Zhong, Z. Wu, Z. Chen, Q. Ren, and J. Zhou, \u0026quot;Advances in the interaction between endoplasmic reticulum stress and osteoporosis,\u0026quot; \u003cem\u003eBiomedicine \u0026amp; Pharmacotherapy, \u003c/em\u003evol. 165, p. 115134, 2023/09/01/ 2023.\u003c/li\u003e\n\u003cli\u003eZ. Jin and W. S. El-Deiry, \u0026quot;Overview of cell death signaling pathways,\u0026quot; \u003cem\u003eCancer Biology \u0026amp; Therapy, \u003c/em\u003evol. 4, no. 2, pp. 147-171, 2005/02/02 2005.\u003c/li\u003e\n\u003cli\u003eT. Anelli and R. Sitia, \u0026quot;Protein quality control in the early secretory pathway,\u0026quot; \u003cem\u003eThe EMBO Journal, \u003c/em\u003evol. 27, no. 2, pp. 315-327, 2008/01/23 2008.\u003c/li\u003e\n\u003cli\u003eA. S. Lee, \u0026quot;The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress,\u0026quot; (in eng), \u003cem\u003eMethods, \u003c/em\u003evol. 35, no. 4, pp. 373-81, 2005.\u003c/li\u003e\n\u003cli\u003eN. T. Sprenkle, S. G. Sims, C. L. S\u0026aacute;nchez, and G. P. Meares, \u0026quot;Endoplasmic reticulum stress and inflammation in the central nervous system,\u0026quot; \u003cem\u003eMolecular Neurodegeneration, \u003c/em\u003evol. 12, no. 1, p. 42, 2017/05/25 2017.\u003c/li\u003e\n\u003cli\u003eS. Chakravarthi, C. E. Jessop, and N. J. Bulleid, \u0026quot;The role of glutathione in disulphide bond formation and endoplasmic‐reticulum‐generated oxidative stress,\u0026quot; \u003cem\u003eEMBO reports, \u003c/em\u003evol. 7, no. 3, pp. 271-275-275, 2006/03/01 2006.\u003c/li\u003e\n\u003cli\u003eI. B. Perez and P. J. Brown, \u0026quot;The role of ROS signaling in cross-tolerance: from model to crop,\u0026quot; (in English), \u003cem\u003eFrontiers in Plant Science, \u003c/em\u003eMini Review vol. 5, 2014-December-23 2014.\u003c/li\u003e\n\u003cli\u003eY. Zhang, S. Choksi, K. Chen, Y. Pobezinskaya, I. Linnoila, and Z.-G. Liu, \u0026quot;ROS play a critical role in the differentiation of alternatively activated macrophages and the occurrence of tumor-associated macrophages,\u0026quot; \u003cem\u003eCell Research, \u003c/em\u003evol. 23, no. 7, pp. 898-914, 2013.\u003c/li\u003e\n\u003cli\u003eY. Che, Y. Tian, R. Chen, L. Xia, F. Liu, and Z. Su, \u0026quot;IL-22 ameliorated cardiomyocyte apoptosis in cardiac ischemia/reperfusion injury by blocking mitochondrial membrane potential decrease, inhibiting ROS and cytochrome C,\u0026quot; \u003cem\u003eBiochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, \u003c/em\u003evol. 1867, no. 9, 2021.\u003c/li\u003e\n\u003cli\u003eQ. Zhang\u003cem\u003e et al.\u003c/em\u003e, \u0026quot;Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress,\u0026quot; \u003cem\u003eApoptosis, \u003c/em\u003evol. 21, no. 4, pp. 432-442, 2016/04/01 2016.\u003c/li\u003e\n\u003cli\u003eY. Tian, L. Wang, Z. Qiu, Y. Xu, and R. Hua, \u0026quot;Autophagy triggers endoplasmic reticulum stress and C/EBP homologous protein-mediated apoptosis in OGD/R-treated neurons in a caspase-12-independent manner,\u0026quot; (in eng), \u003cem\u003eJournal of neurophysiology, \u003c/em\u003evol. 126, no. 5, pp. 1740-1750, 2021/11// 2021.\u003c/li\u003e\n\u003cli\u003eH. Wootz, I. Hansson, L. Korhonen, U. N\u0026auml;p\u0026auml;nkangas, and D. Lindholm, \u0026quot;Caspase-12 cleavage and increased oxidative stress during motoneuron degeneration in transgenic mouse model of ALS,\u0026quot; \u003cem\u003eBiochemical and Biophysical Research Communications, \u003c/em\u003evol. 322, no. 1, pp. 281-286, 2004/09/10/ 2004.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"patulin, fisetin, myocardial damage, Grp78, Chop, Caspase-12","lastPublishedDoi":"10.21203/rs.3.rs-4839276/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4839276/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjectives of the Study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFisetin (FIS) has a good protective effect on the heart. However, fisetin in regulating the role of the myocardial injury induced by patulin (PAT) is not clear. The aim of this study is to investigate the possible mechanism of fisetin in attenuating patulin induced myocardial injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCardiomyocytes were treated with 25μM PAT to set the control group, FIS only group, PAT only group and PAT-FIS addition group. LDH activity, SOD content, and MDA content were evaluated using kits. ROS levels were determined by measuring the intensity of fluorescence. Mitochondrial membrane potential was detected by JC-1 dye staining. The protein expressions of Grp78, Chop and Caspase-12 were detected by Western blot.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn PAT-FIS group, LDH release and MDA content decreased, but SOD content increased. Compared with the control group, the level of ROS in PAT group increased more than 10 times. The level of ROS in the PAT-FIS group was still higher than that in the control group, but it was significantly lower than that in the PAT group. The proportion of red fluorescence in the mitochondrial membrane potential of cardiomyocytes increased from 75% to 85% in the PAT-FIS group. PAT up-regulated the expression of Chop, Grp78 and Caspase-12 proteins, while the overexpression of Chop, Grp78 and Caspase-12 proteins was inhibited after pretreatment with FIS and PAT .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur findings suggest that FIS inhibits PAT-induced cardiomyocyte apoptosis by regulating ROS/Grp78/Chop/Caspase-12 signaling.\u003c/p\u003e","manuscriptTitle":"Fisetin inhibits patulin-induced cardiomyocyte apoptosis by regulating ROS/Grp78/Chop/Caspase-12","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-30 18:54:12","doi":"10.21203/rs.3.rs-4839276/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8f56e5ef-8020-4c85-8632-a84f2bbdae1f","owner":[],"postedDate":"August 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-04T10:34:34+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-30 18:54:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4839276","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4839276","identity":"rs-4839276","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}